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公开(公告)号：US07696337B2
公开(公告)日：2010-04-13
申请号：US11574307
申请日：2005-08-26
申请人： Michael M. Becker , Steven T. Brentano , Daniel P. Kolk , Wai-Chung Lam , Kristin W. Livezey , Norman C. Nelson , Astrid R. W. Schroder , Gary P. Schroth
发明人： Michael M. Becker , Steven T. Brentano , Daniel P. Kolk , Wai-Chung Lam , Kristin W. Livezey , Norman C. Nelson , Astrid R. W. Schroder , Gary P. Schroth
CPC分类号： C12P19/34 , C12Q1/6865 , C12Q2537/163 , C12Q2521/325 , C12Q2521/107 , C12Q2525/186 , C12Q2533/101 , C12Q2525/143
摘要： The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present invention discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side products. The method uses only one primer, the “priming oligonucleotide,” a 3′blocked promoter oligonucleotide and optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or eliminating the formation of side products. The method of the present invention minimizes or eliminates the emergence of side products, thus providing a high level of specificity. Furthermore, the appearance of side products can complicate the analysis of the amplification reaction by various molecular detection techniques. The present invention minimizes or eliminates this problem, thus providing an enhanced level of sensitivity.
摘要翻译： 本发明涉及合成目标核酸序列的多个拷贝的新型方法，所述目标核酸序列是自动催化的（即，能够自动循环而不需要修饰诸如温度，pH或离子强度的反应条件，并且使用一种 循环在下一个）。 特别地，本发明公开了一种鲁棒有效的核酸扩增方法，同时减少了副产物的外观。 该方法仅使用一种引物，即引物寡核苷酸，3'封闭的启动子寡核苷酸和任选的终止引物延伸反应的手段，以体外扩增RNA或DNA分子，同时减少或消除副产物的形成。 本发明的方法使副产物的出现最小化或消除，从而提供高水平的特异性。 此外，副产物的出现可能通过各种分子检测技术使扩增反应的分析复杂化。 本发明使这个问题最小化或消除了这一点，从而提供了增强的灵敏度。
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公开(公告)号：US07374885B2
公开(公告)日：2008-05-20
申请号：US11213519
申请日：2005-08-26
申请人： Michael M. Becker , Wai-Chung Lam , Kristin W. Livezey , Steven T. Brentano , Daniel P. Kolk , Astrid R. W. Schroder
发明人： Michael M. Becker , Wai-Chung Lam , Kristin W. Livezey , Steven T. Brentano , Daniel P. Kolk , Astrid R. W. Schroder
CPC分类号： C12P19/34 , C12Q1/6865 , C12Q2537/163 , C12Q2521/325 , C12Q2521/107 , C12Q2525/186 , C12Q2533/101 , C12Q2525/143
摘要： The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present invention discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side-products. The method uses only one primer, the “priming oligonucleotide,” a promoter oligonucleotide modified to prevent polymerase extension from its 3′-terminus and, optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or substantially eliminating the formation of side-products. The method of the present invention minimizes or substantially eliminates the emergence of side-products, thus providing a high level of specificity. Furthermore, the appearance of side-products can complicate the analysis of the amplification reaction by various molecular detection techniques. The present invention minimizes or substantially eliminates this problem, thus providing an enhanced level of sensitivity.
摘要翻译： 本发明涉及合成目标核酸序列的多个拷贝的新型方法，所述目标核酸序列是自动催化的（即，能够自动循环而不需要修饰诸如温度，pH或离子强度的反应条件，并且使用一种 循环在下一个）。 特别地，本发明公开了一种鲁棒有效的核酸扩增方法，同时减少副产物的外观。 该方法仅使用一种引物，即引物寡核苷酸，经修饰的启动子寡核苷酸，用于防止聚合酶从其3'末端延伸，以及任选的终止引物延伸反应的手段，以在体外扩增RNA或DNA分子，同时 减少或基本上消除副产物的形成。 本发明的方法使副产物的出现最小化或基本上消除，从而提供高水平的特异性。 此外，副产物的出现可能通过各种分子检测技术使扩增反应的分析复杂化。 本发明使这个问题最小化或基本消除，从而提供了增强的灵敏度。
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公开(公告)号：US07939260B2
公开(公告)日：2011-05-10
申请号：US12389993
申请日：2009-02-20
申请人： Michael M. Becker , Steven T. Brentano , Kristin W. Livezey , Norman C. Nelson , Gary P. Schroth
发明人： Michael M. Becker , Steven T. Brentano , Kristin W. Livezey , Norman C. Nelson , Gary P. Schroth
CPC分类号： C12P19/34 , C12Q1/6865 , C12Q2537/163 , C12Q2521/325 , C12Q2521/107 , C12Q2525/186 , C12Q2533/101 , C12Q2525/143
摘要： The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present invention discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side-products. The method uses only one primer, the “priming oligonucleotide,” a promoter oligonucleotide modified to prevent polymerase extension from its 3′-terminus and, optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or substantially eliminating the formation of side-products. The method of the present invention minimizes or substantially eliminates the emergence of side-products, thus providing a high level of specificity. Furthermore, the appearance of side-products can complicate the analysis of the amplification reaction by various molecular detection techniques. The present invention minimizes or substantially eliminates this problem, thus providing an enhanced level of sensitivity.
摘要翻译： 本发明涉及合成目标核酸序列的多个拷贝的新型方法，所述目标核酸序列是自动催化的（即，能够自动循环而不需要修饰诸如温度，pH或离子强度的反应条件，并且使用一种 循环在下一个）。 特别地，本发明公开了一种鲁棒有效的核酸扩增方法，同时减少副产物的外观。 该方法仅使用一种引物，即引物寡核苷酸，经修饰的启动子寡核苷酸，用于防止聚合酶从其3'末端延伸，以及任选的终止引物延伸反应的手段，以在体外扩增RNA或DNA分子，同时 减少或基本上消除副产物的形成。 本发明的方法使副产物的出现最小化或基本上消除，从而提供高水平的特异性。 此外，副产物的出现可能通过各种分子检测技术使扩增反应的分析复杂化。 本发明使这个问题最小化或基本消除，从而提供了增强的灵敏度。
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4.发明申请公开(公告)号：US20120003651A1
公开(公告)日：2012-01-05
申请号：US13231848
申请日：2011-09-13
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6848 , C12N15/1065 , C12Q1/6806 , C12Q1/689 , C12Q1/6895 , C12Q2525/155
摘要： The present invention provides nucleic acid amplification methods that desirably reduce or eliminate false positive amplification signals resulting from contaminating biological material, e.g., nucleic acid, that may be present in one or more reagents used in an amplification reaction and/or that may be present in the environment in which an amplification reaction is performed. The invention offers the further advantage of requiring less stringent purification and/or sterility efforts than conventionally needed in order to ensure that enzymes and other reagents used in amplification reactions, and the environment in which an amplification reaction is performed, are free of bacterial or other nucleic acid contamination that may yield false positive results.
摘要翻译： 本发明提供了核酸扩增方法，其期望地减少或消除由污染生物材料（例如核酸）产生的假阳性扩增信号，所述核酸可以存在于用于扩增反应的一种或多种试剂中和/或可存在于 进行扩增反应的环境。 本发明提供了进一步的优点，即需要比常规所需的更少的纯化和/或不育努力，以确保用于扩增反应的酶和其它试剂以及进行扩增反应的环境不含细菌或其它 可能产生假阳性结果的核酸污染物。
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公开(公告)号：US10316352B2
公开(公告)日：2019-06-11
申请号：US12465323
申请日：2009-05-13
IPC分类号： C12Q1/68 , C07H21/00 , C12Q1/6816 , C12Q1/6832 , C12Q1/6834 , C12Q1/686
摘要： The present invention provides compositions, kits and methods for the selective hybridization and capture of a specific target nucleic acid. The specific target nucleic acid may be present in a heterogeneous mixture of nucleic acids. Selective hybridization and capture provides a target nucleic acid that is substantially free of non-target and/or contaminating nucleic acids. Target nucleic acids that have been selectively hybridized and captured using the current invention are then used in subsequent analysis, wherein the presence of non-target and/or contaminating nucleic acids that interfere with said subsequent analysis have been substantially reduced or eliminated, thereby providing improved analysis results. The invention offers the further advantage of requiring less stringent purification and/or sterility efforts than conventionally needed in order to ensure that enzymes and other reagents used in subsequent analysis, or present in the environment in which an assay is performed, are free of bacterial or other contaminating nucleic acids.
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6.发明授权公开(公告)号：US08278052B2
公开(公告)日：2012-10-02
申请号：US13231848
申请日：2011-09-13
CPC分类号： C12Q1/6848 , C12N15/1065 , C12Q1/6806 , C12Q1/689 , C12Q1/6895 , C12Q2525/155
摘要： The present invention provides kits containing tagged oligonucleotides for use in certain nucleic acid amplification methods to desirably reduce or eliminate false positive amplification signals resulting from contaminating biological material, e.g., nucleic acid, that may be present in one or more reagents used in an amplification reaction and/or that may be present in the environment in which an amplification reaction is performed. The kits containing tagged oligonucleotides can be used in purification and/or sterility efforts under less stringent conditions than conventionally needed to reduce or eliminate false positive results in a nucleic acid amplification method.
摘要翻译： 本发明提供了含有用于某些核酸扩增方法中的标记的寡核苷酸的试剂盒，用于期望地减少或消除由可能存在于用于扩增反应的一种或多种试剂中的生物材料例如核酸引起的假阳性扩增信号 和/或其可以存在于进行扩增反应的环境中。 含有标记的寡核苷酸的试剂盒可以在比通常在减少或消除核酸扩增方法中的假阳性结果所需的不严格条件下用于纯化和/或不育的努力。
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公开(公告)号：US08183359B2
公开(公告)日：2012-05-22
申请号：US12716052
申请日：2010-03-02
IPC分类号： C07H21/04
CPC分类号： C12P19/34 , C12Q1/6865 , C12Q2533/101 , C12Q2525/186 , C12Q2525/143
摘要： Kits for amplifying DNA which include a priming oligonucleotide that hybridizes to a 3′-end of a DNA target sequence, a displacer oligonucleotide that hybridizes to a target nucleic acid containing the DNA target sequence at a position upstream from the priming oligonucleotide, and a promoter oligonucleotide that includes a region that hybridizes to a 3′-region of a DNA primer extension product that includes the priming oligonucleotide and a promoter for an RNA polymerase. The priming oligonucleotide does not include an RNA region that hybridizes to the target nucleic acid and is selectively degraded by an enzyme activity when hybridized to the target nucleic acid. The kits do not include a restriction endonuclease and oligonucleotides that include a promoter for an RNA polymerase are all modified to prevent the initiation of DNA synthesis therefrom.
摘要翻译： 用于扩增DNA的试剂盒，其包含与DNA靶序列的3'端杂交的引物寡核苷酸，与引物寡核苷酸上游位置含有DNA靶序列的靶核酸杂交的置换者寡核苷酸，以及启动子 包括与包含引发寡核苷酸的DNA引物延伸产物的3'区域和RNA聚合酶的启动子杂交的区域的寡核苷酸。 引发寡核苷酸不包括与靶核酸杂交的RNA区域，并且当与靶核酸杂交时被选择性地被酶活性降解。 试剂盒不包括限制性内切核酸酶，并且包括RNA聚合酶的启动子的寡核苷酸都被修饰以防止从其中开始DNA合成。
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8.发明授权公开(公告)号：US08034570B2
公开(公告)日：2011-10-11
申请号：US12892323
申请日：2010-09-28
CPC分类号： C12Q1/6848 , C12N15/1065 , C12Q1/6806 , C12Q1/689 , C12Q1/6895 , C12Q2525/155
摘要： Disclosed are methods for selective amplification of target nucleic acid sequence using a tagged oligonucleotide comprising a target hybridizing sequence that hybridizes to a 3′-end of the target nucleic acid and a tag sequence situated 5′ to the target hybridizing sequence. The tagged oligonucleotide is hybridized to the target nucleic acid and, after reducing the effective concentration of unhybridized tagged oligonucleotide having an active form, a primer extension reaction is initiated to produce a primer extension product. Further amplification also utilizes a first oligonucleotide that hybridizes to the 3′ end of the complement of the target nucleic acid and a second oligonucleotide that hybridizes to the complement of the tag sequence. Also disclosed are reaction mixtures for use in the disclosed methods comprising the tagged oligonucleotide hybridized to target nucleic and substantially free of an active form of unhybridized tagged oligonucleotide.
摘要翻译： 公开了使用包含与靶核酸的3'-末端杂交的靶杂交序列和位于靶标杂交序列5'的标签序列的标记寡核苷酸选择性扩增靶核酸序列的方法。 标记的寡核苷酸与靶核酸杂交，并且在降低具有活性形式的未杂交标记的寡核苷酸的有效浓度之后，引发引物延伸反应以产生引物延伸产物。 进一步扩增还利用与靶核酸的补体的3'端杂交的第一寡核苷酸和与标签序列的互补杂交的第二寡核苷酸。 还公开了用于所公开方法的反应混合物，其包含与靶核酸杂交的基本上不含活性形式的未杂交标记的寡核苷酸的标记寡核苷酸。
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9.发明授权公开(公告)号：US08580510B2
公开(公告)日：2013-11-12
申请号：US13612601
申请日：2012-09-12
CPC分类号： C12Q1/6848 , C12N15/1065 , C12Q1/6806 , C12Q1/689 , C12Q1/6895 , C12Q2525/155
摘要： The present invention provides nucleic acid amplification systems and methods that desirably reduce or eliminate false positive amplification signals resulting from contaminating biological material, e.g., nucleic acid, that may be present in one or more reagents used in an amplification reaction and/or that may be present in the environment in which an amplification reaction is performed. The invention offers the further advantage of requiring less stringent purification and/or sterility efforts than conventionally needed in order to ensure that enzymes and other reagents used in amplification reactions, and the environment in which an amplification reaction is performed, are free of bacterial or other nucleic acid contamination that may yield false positive results.
摘要翻译： 本发明提供了核酸扩增系统和方法，其期望地减少或消除由可能存在于扩增反应中使用的一种或多种试剂中的生物材料例如核酸引起的假阳性扩增信号和/或可能是 存在于进行扩增反应的环境中。 本发明提供了进一步的优点，即需要比常规所需的更少的纯化和/或不育努力，以确保用于扩增反应的酶和其它试剂以及进行扩增反应的环境不含细菌或其它 可能产生假阳性结果的核酸污染物。
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10.发明授权公开(公告)号：US07833716B2
公开(公告)日：2010-11-16
申请号：US11810834
申请日：2007-06-06
CPC分类号： C12Q1/6848 , C12N15/1065 , C12Q1/6806 , C12Q1/689 , C12Q1/6895 , C12Q2525/155
摘要： A method for selective amplification of at least one target nucleic acid sequence, comprising the steps of: treating a sample with a tagged oligonucleotide comprising a target hybridizing sequence that hybridizes to a 3′-end of the target nucleic acid sequence, and a tag sequence situated 5′ to the target hybridizing sequence that does not stably hybridize to a target nucleic acid, wherein tagged oligonucleotide hybridized to target nucleic acids form tagged target nucleic acids; prior to initiating a primer extension reaction, reducing the effective concentration of unhybridized tagged oligonucleotide having an active form; initiating an extension reaction to produce a primer extension product; separating the primer extension product from the target nucleic acid; and producing amplification products therefrom using an oligonucleotide that hybridizes to the complement of the tag sequence.
摘要翻译： 一种用于选择性扩增至少一种靶核酸序列的方法，包括以下步骤：用包含与靶核酸序列的3'-末端杂交的靶杂交序列的标记寡核苷酸处理样品，以及标签序列 位于与靶核酸不稳定杂交的靶杂交序列5'处，其中与靶核酸杂交的标记的寡核苷酸形成标记的靶核酸; 在引发引物延伸反应之前，降低具有活性形式的未杂交标记的寡核苷酸的有效浓度; 引发延伸反应以产生引物延伸产物; 将引物延伸产物与靶核酸分离; 并使用与标签序列的互补体杂交的寡核苷酸从其产生扩增产物。
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